Lentiviral vectors have many features of potential value to the development of vectors for CF gene therapy. Experiments in a number of other organ systems have demonstrated the ability of lentiviral vectors for efficiently transducing nondividing cells. In most cases, transgene expression is permanent due to the integration of the vector genome. Specific applications of lentiviral vectors for gene transfer to the airway have been unsuccessful due in part to insufficient uptake of the vector. Most studies have utilized ientiviral vector pseudotypes which have incorporated the envelope protein of vesticular stomatitis virus whose receptor does not appear to be accessible to vector administered into the lumen of the airway. In an attempt to overcome this block in entry, we undertook a series of experiments to evaluate lentiviral vectors pseudotyped with envelopes from other viruses. These studies demonstrate the ability of lentiviral vector pseudotyped with the envelope protein from Ebola Zaire (EboZ) to efficiently target airway epithelial cells via the apical surface. The goal of this project is to build on this initial success to develop safe and efficient lentiviral vectors for in vivo gene therapy. This will require the creation of pseudotypes with other envelopes as well as characterization of the host responses to vector in terms of immunology and insertional mutagenesis. The Specific Aims are as follows. 1) Alternative vector pseudotypes will be developed utilizing envelopes from other viruses as well as envelope chimeras. The most promising constructs will be evaluated for in vivo gene transfer in mice and nonhuman primates. 2) it is presumed that antigen specific immune responses to in vivo gene therapy will impact on the utility of lentiviral vectors which has not been thoroughly evaluated in any system. We will determine the B and T cell responses following in vivo delivery of lentiviruses and assess their impact on the utility of this approach. 3) A critical aspect of assessing the safety of lentiviral vectors is the potential for insertional mutagenesis. The molecular state of the vector genome will be evaluated with respect to the existence of integrated vs. nonintegrated forms as well as the distribution of proviruses that integrate.